The invention relates to improved vectors, and more specifically, improved adenovirus vectors useful for gene therapy.
Adenoviruses (Ad) are double-stranded DNA viruses. The genome of adenoviruses (xcx9c36 kb) is complex and contains over 50 open reading frames (ORFs). These ORFs are overlapping and genes encoding one protein are often embedded within genes coding for other Ad proteins. Expression of Ad genes is divided into an early and a late phase. Early genes are those transcribed prior to replication of the genome while late genes are transcribed after replication. The early genes comprise E1a, E1b, E2a, E2b, E3 and E4. The E1a gene products are involved in transcriptional regulation; the E1b gene products are involved in the shut-off of host cell functions and MRNA transport. E2a encodes the a DNA-binding protein (DBP); E2b encodes the viral DNA polymerase and preterminal protein (pTP). The E3 gene products are not essential for viral growth in cell culture. The E4 region encodes regulatory protein involved in transcriptional and post-transcriptional regulation of viral gene expression; a subset of the E4 proteins are essential for viral growth. The products of the late genes (e.g., L1-5) are predominantly components of the virion as well as proteins involved in the assembly of virions. The VA genes produce VA RNAs which block the host cell from shutting down viral protein synthesis.
Adenoviruses or Ad vectors have been exploited for the delivery of foreign genes to cells for a number of reasons including the fact that Ad vectors have been shown to be highly effective for the transfer of genes into a wide variety of tissues in vivo and the fact that Ad infects both dividing and non-dividing cells; a number of tissues which are targets for gene therapy comprise largely non-dividing cells.
The current generation of Ad vectors suffer from a number of limitations which preclude their widespread clinical use. The most serious limitation is the loss of expression of genes of interest in cells infected with Ad vectors. It has been assumed that this loss in expression is due to immune detection and elimination of cells infected with Ad vectors, but more recently transcriptional regulation has been raised as a potential factor in loss of transgene expression.
What is needed is an approach that overcomes the problem of loss of expression of genes of interest in cells infected with Ad vectors. Such an approach should ensure long-term expression for gene therapy and other applications.
The present invention contemplates improving vectors generally, and more specifically improving adenovirus vectors. The present invention contemplates both improved compositions (e.g., expression vectors) and methods (e.g., methods of transfection and gene therapy). With regard to compositions, the present invention contemplates an expression vector comprising a ribosomal promoter sequence operably linked to a gene of interest. The invention is not limited by the nature of the ribosomal promoter sequence chosen; any non-viral promoter sequence or portion thereof which is functional in cells (i.e., such that a gene of interest can be expressed and/or overexpressed) may be utilized. A variety of ribosomal promoters are known to those skilled in the art. Preferred ribosomal promoters are eukaryotic ribosomal promoters, including but not limited to mammalian ribosomal promoters such as those of mice, rats, rabbits, pigs and humans.
In a preferred embodiment, said expression vector further comprises viral nucleic acid. It is not intended that the expression vector be limited to a particular viral vector. In one embodiment, said expression vector further comprises adenoviral nucleic acid. Indeed, the present invention contemplates replication-defective adenoviral vectors comprising a ribosomal promoter sequence operably linked to a genetic cassette encoding one or more gene products. In a preferred embodiment, adenoviral vectors shown to be free of E1 function (e.g., by absence of replication on HeLA cells) are contemplated, such vectors comprising a ribosomal promoter sequence operably linked to a genetic cassette encoding one or more gene products.
The present invention also contemplates a mammalian cell line containing the above-described recombinant vector and integrated viral sequences expressing E1 function. It is preferred that said cell line is a 293-derived cell line.
With regard to methods of transfection, the present invention contemplates a method, comprising: a) providing: i) eukaryotic cells, ii) an expression vector comprising a ribosomal promoter sequence operably linked to a genetic cassette encoding one or more gene products; and b) introducing said expression vector into said cells. Again, in a preferred embodiment, said expression vector further comprises viral nucleic acid, such as adenoviral nucleic acid.
With regard to methods of gene therapy, in one embodiment, the present invention contemplates a method for delivering nucleic acid to cells of an animal, comprising: a) providing: i) an expression vector comprising a ribosomal promoter sequence operably linked to a gene of interest, ii) a recipient animal; and b) administering said vector to said recipient animal.
In a particular embodiment, the oligonucleotide or gene cassette is delivered to a particular tissue in said animal. It is not intended that the present invention be limited to the particular tissue type. In one embodiment, however, said tissue is selected from the group consisting of lung, trachea and liver tissue. For delivery to the liver, the hepatocytes can be readily transfected in vivo by direct vector infusion in the portal vein, as well as to the peripheral circulation.
With regard to other methods of gene therapy, in another embodiment, the present invention contemplates a method for delivering nucleic acid to cells of an animal, comprising: a) providing: i) an expression vector comprising a ribosomal promoter sequence operably linked to a gene of interest, ii) a recipient animal; b) coupling said expression vector to a carrier to generate a composition; and c) administering said composition to said recipient animal.
Where adenoviral vectors are employed in the present invention it is not intended that the present invention be limited by the precise size of the vector, although it is generally desirable that the vector have a total size of between 20 and 40 kilobase pairs. It is preferred that the total size of the DNA packaged into an adenovirus particle derived from these vectors is about the length of the wild-type adenovirus genome (xcx9c36 kb). It is well known in the art that DNA representing about 105% of the wild-type length may be packaged into a viral particle; thus the adenovirus particle derived from recombinant vector may contain DNA whose length exceeds by xcx9c105% the size of the wild-type genome. The size of the recombinant plasmid may be adjusted using reporter genes and genes of interest having various sizes (including the use of different sizes of introns within these genes) as well as through the use of irrelevant or non-coding DNA fragment which act as xe2x80x9cstufferxe2x80x9d fragments (e.g., portions of bacteriophage genomes).
In one embodiment, the present invention contemplates recovering said encapsidated adenovirus minichromosome and, in turn, purifying said recovered encapsidated adenovirus minichromosome. Thereafter, said purified encapsidated adenovirus minichromosome can be administered to a host (e.g. a mammal). Human therapy is thereby contemplated.
It is not intended that the present invention be limited by the nature of the administration of said adenovirus minichromosomes. All types of administration are contemplated, including direct injection (intramuscular, intravenous, subcutaneous, etc.), inhalation, etc.
FIG. 1 shows mouse L32 ribosomal promoter sequences (SEQ ID NO:1).
FIG. 2 shows pig ribosomal promoter sequences (SEQ ID NO:2).
FIG. 3 shows the results of in vivo transfection of liver of recipient animals (with a vector comprising a viral promoter) by Southern analysis.
FIG. 4 shows a restriction map of an adenoviral vector comprising a ribosomal promoter in operable combination with a gene of interest. The plasmid is 9.30 kb; xe2x80x9camp rxe2x80x9d refers to the ampicillin resistance gene and xe2x80x9cbacterial orixe2x80x9d refers to the bacterial origin of replication. Human adenovirus type 5 sequences are denoted as map units (m.u.; 1 m.u.=360 bp).
FIG. 5 shows the results of in vivo transfection of liver of recipient animals (with a vector comprising a ribosomal promoter) by Northern analysis.
FIGS. 6A and 6B shows the bilirubin levels of control (Panel B) and treated (Panel A) animals following transfection with a vector comprising a ribosomal promoter.
FIGS. 7A-C is a graph showing HPLC analysis of bile for bilirubin glucuronides in untreated animals (A), and animals transfected with the transferase gene (B) or a reporter gene (C).
FIG. 8 shows the results of in vivo transfection of liver of recipient animals (with a vector comprising a ribosomal promoter) by Southern analysis.
To facilitate understanding of the invention, a number of terms are defined below.
The term xe2x80x9cgenexe2x80x9d refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor thereof. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired enzymatic activity is retained. The term xe2x80x9cgenexe2x80x9d encompasses both cDNA and genomic forms of a given gene.
The term xe2x80x9cwild-typexe2x80x9d refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the xe2x80x9cnormalxe2x80x9d or xe2x80x9cwild-typexe2x80x9d form of the gene. In contrast, the term xe2x80x9cmodifiedxe2x80x9d or xe2x80x9cmutantxe2x80x9d refers to a gene or gene product which displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
The term xe2x80x9coligonucleotidexe2x80x9d as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, usually more than three (3), and typically more than ten (10) and up to one hundred (100) or more (although preferably between twenty and thirty). The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.
As used herein, the term xe2x80x9cregulatory elementxe2x80x9d refers to a genetic element which controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc. (defined below).
Transcriptional control signals in eucaryotes comprise xe2x80x9cpromoterxe2x80x9d and xe2x80x9cenhancerxe2x80x9d elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription [Maniatis, T. et al., Science 236:1237 (1987)]. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in procaryotes).
The term xe2x80x9cheterologous DNA sequencexe2x80x9d refers to a nucleotide sequence which is not endogenous to the cell into which it is introduced. Heterologous DNA includes a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA also includes a nucleotide sequence which is naturally found in the cell into which it is introduced and which contains some modification relative to the naturally-occurring sequence. An example of heterologous DNA of the present invention comprises a heterologous regulatory sequence such as a heterologous ribosomal promoter which is not found in the mammalian cell into which it is introduced. However, the present invention also contemplates endogenous (also called xe2x80x9chomologousxe2x80x9d) ribosomal promoters in operable combination with heterologous genes of interest.
The term xe2x80x9crecombinant DNA vectorxe2x80x9d as used herein refers to DNA sequences containing a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., mammal). DNA sequences necessary for expression in procaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, polyadenlyation signals and enhancers.
The terms xe2x80x9cin operable combinationxe2x80x9d, xe2x80x9cin operable orderxe2x80x9d and xe2x80x9coperably linkedxe2x80x9d as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
The term xe2x80x9cgenetic cassettexe2x80x9d as used herein refers to a fragment or segment of DNA containing a particular grouping of genetic elements. The cassette can be removed and inserted into a vector or plasmid as a single unit. A xe2x80x9cplasmid backbonexe2x80x9d refers to a piece of DNA containing at least plasmid origin of replication and a selectable marker gene (e.g., an antibiotic resistance gene) which allows for selection of bacterial hosts containing the plasmid; the plasmid backbone may also include a polylinker region to facilitate the insertion of genetic elements within the plasmid. When a particular plasmid is modified to contain non-plasmid elements (e.g., insertion of Ad sequences and/or a eukaryotic gene of interest linked to a ribosomal promoter), the plasmid sequences are referred to as the plasmid backbone.
Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5xe2x80x2 phosphate-of one mononucleotide pentose ring is attached to the 3xe2x80x2 oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the xe2x80x9c5xe2x80x2 endxe2x80x9d if its 5xe2x80x2 phosphate is not linked to the 3xe2x80x2 oxygen of a mononucleotide pentose ring and as the xe2x80x9c3xe2x80x2 endxe2x80x9d if its 3xe2x80x2 oxygen is not linked to a 5xe2x80x2 phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5xe2x80x2 and 3xe2x80x2 ends.
When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3xe2x80x2 end of one oligonucleotide points towards the 5xe2x80x2 end of the other, the former may be called the xe2x80x9cupstreamxe2x80x9d oligonucleotide and the latter the xe2x80x9cdownstreamxe2x80x9d oligonucleotide.
The term xe2x80x9cprimerxe2x80x9d refers to an oligonucleotide which is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated. An oligonucleotide xe2x80x9cprimerxe2x80x9d may occur naturally, as in a purified restriction digest or may be produced synthetically.
A primer is selected to be xe2x80x9csubstantiallyxe2x80x9d complementary to a strand of specific sequence of the template. A primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5xe2x80x2 end of the primer, with the remainder of the primer sequence being substantially complementary to the strand. Non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize and thereby form a template primer complex for synthesis of the extension product of the primer.
xe2x80x9cHybridizationxe2x80x9d methods involve the annealing of a complementary sequence to the target nucleic acid (the sequence to be detected). The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon. The initial observations of the xe2x80x9chybridizationxe2x80x9d process by Marmur and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960) have been followed by the refinement of this process into an essential tool of modem biology.
The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5xe2x80x2 end of one sequence is paired with the 3xe2x80x2 end of the other, is in xe2x80x9cantiparallel association.xe2x80x9d Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.
Stability of a nucleic acid duplex is measured by the melting temperature, or xe2x80x9cTm.xe2x80x9d The Tm of a particular nucleic acid duplex under specified conditions is the temperature at which on average half of the base pairs have disassociated. The equation for calculating the Tm of nucleic acids is well known in the art.
The term xe2x80x9cprobexe2x80x9d as used herein refers to a labeled oligonucleotide which forms a duplex structure with a sequence in another nucleic acid, due to complementarity of at least one sequence in the probe with a sequence in the other nucleic acid.
The term xe2x80x9clabelxe2x80x9d as used herein refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like.
The terms xe2x80x9cnucleic acid substratexe2x80x9d and nucleic acid templatexe2x80x9d are used herein interchangeably and refer to a nucleic acid molecule which may comprise single- or double-stranded DNA or RNA.
xe2x80x9cOligonucleotide primers matching or complementary to a gene sequencexe2x80x9d refers to oligonucleotide primers capable of facilitating the template-dependent synthesis of single or double-stranded nucleic acids. Oligonucleotide primers matching or complementary to a gene sequence may be used in PCRS, RT-PCRs and the like.
A xe2x80x9cconsensus gene sequencexe2x80x9d refers to a gene sequence which is derived by comparison of two or more gene sequences and which describes the nucleotides most often present in a given segment of the genes; the consensus sequence is the canonical sequence.
The term xe2x80x9cpolymorphic locusxe2x80x9d is a locus present in a population which shows variation between members of the population (i.e., the most common allele has a frequency of less than 0.95). In contrast, a xe2x80x9cmonomorphic locusxe2x80x9d is a genetic locus at little or no variations seen between members of the population (generally taken to be a locus at which the most common allele exceeds a frequency of 0.95 in the gene pool of the population).
The term xe2x80x9cmicroorganismxe2x80x9d as used herein means an organism too small to be observed with the unaided eye and includes, but is not limited to bacteria, viruses, protozoans, fungi, and ciliates.
The term xe2x80x9cmicrobial gene sequencesxe2x80x9d refers to gene sequences derived from a microorganism.
The term xe2x80x9cbacteriaxe2x80x9d refers to any bacterial species including eubacterial and archaebacterial species.
The term xe2x80x9cvirusxe2x80x9d refers to obligate, ultramicroscopic, intracellular parasites incapable of autonomous replication (i.e., replication requires the use of the host cell""s machinery). Adenoviruses, as noted above, are double-stranded DNA viruses. The left and right inverted terminal repeats (ITRs) are short elements located at the 5xe2x80x2 and 3xe2x80x2 termini of the linear Ad genome, respectively and are required for replication of the viral DNA. The left ITR is located between 1-130 bp in the Ad genome (also referred to as 0-0.5 mu). The right ITR is located from xcx9c3,7500 bp to the end of the genome (also referred to as 99.5-100 mu). The two ITRs are inverted repeats of each other. The 5xe2x80x2 end of the left ITR is located at the extreme 5xe2x80x2 end of the linear adenoviral genome; picturing the left ITR (LITR) as an arrow extending from the 5xe2x80x2 end of the genome, the tail of the 5xe2x80x2 ITR is located at mu 0 and the head of the left ITR is located at xcx9c0.5 mu (further the tail of the left ITR is referred to as the 5xe2x80x2 end of the left ITR and the head of the left ITR is referred to as the 3xe2x80x2 end of the left ITR). The tail of the right or 3xe2x80x2 ITR is located at mu 100 and the head of the right ITR is located at xcx9cmu 99.5; the head of the right ITR is referred to as the 5xe2x80x2 end of the right ITR and the tail of the right ITR is referred to as the 3xe2x80x2 end of the right ITR (RITE). In the linear Ad genome, the ITRs face each other with the head of each ITR pointing inward toward the bulk of the genome. When arranged in a xe2x80x9ctail to tail orientationxe2x80x9d the tails of each ITR (which comprise the 5xe2x80x2 end of the LITR and the 3xe2x80x2 end of the RITE) are located in proximity to one another while the heads of each ITR are separated and face outward. The xe2x80x9cadenovirus packaging sequencexe2x80x9d refers to the xcexa8 sequence which comprises five (AI-AV) packaging signals and is required for encapsidation of the mature linear genome; the packaging signals are located from xcx9c194 to 358 bp in the Ad genome (about 0.5-1.0 mu).
The phrase xe2x80x9cat least one adenovirus gene coding regionxe2x80x9d refers to a nucleotide sequence containing one or more than one adenovirus gene coding sequence. A xe2x80x9chelper adenovirusxe2x80x9d or xe2x80x9chelper virusxe2x80x9d refers to an adenovirus which is replication-competent in a particular host cell (the host may provide Ad gene products such as E1 proteins), this replication-competent virus is used to supply in trans functions (e.g., proteins) which are lacking in a second replication-incompetent virus; the first replication-competent virus is said to xe2x80x9chelpxe2x80x9d the second replication-incompetent virus thereby permitting the propagation of the second viral genome in the cell containing the helper and second viruses.
The term xe2x80x9ccontaining a deletion within the E1b regionxe2x80x9d refers to a deletion of at least one basepair (preferably more than one bp and preferably at least 100 and most preferably more than 300 bp) within the E1b region of the adenovirus genome. An E1b deletion is a deletion that prevents expression of at least one E1b gene product. In a preferred embodiment, the present invention contemplates a vector wherein the sequences spanning the E1a, E1b and E3b regions are deleted.
An xe2x80x9cadenovirus minichromosomexe2x80x9d refers to a linear molecule of DNA containing the Ad ITRs on each end which is generated from a plasmid containing the ITRs and one or more gene of interest. The term xe2x80x9cencapsidated adenovirus minichromosomexe2x80x9d or xe2x80x9cEAMxe2x80x9d refers to an adenovirus minichromosome which has been packaged or encapsidated into a viral particle. When used herein, xe2x80x9crecoveringxe2x80x9d encapsidated adenovirus minichromosomes refers to the collection of EAMs from a cell containing an EAM plasmid and a helper virus; this cell will direct the encapsidation of the minichromosome to produce EAMs. The EAMs may be recovered from these cells by lysis of the cell (e.g., freeze-thawing) and pelleting of the cell debris to a cell extract. xe2x80x9cPurifyingxe2x80x9d such minichromosomes refers to the isolation of the recovered EAMs in a more concentrated form (relative to the cell lysate) using techniques such as on a density gradient; purification of recovered EAMs permits the physical separation of the EAM from any helper virus (if present).
The term xe2x80x9ctransfectionxe2x80x9d as used herein refers to the introduction of foreign DNA into eukaryotic cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
The term xe2x80x9cstable transfectionxe2x80x9d or xe2x80x9cstably transfectedxe2x80x9d refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term xe2x80x9cstable transfectantxe2x80x9d refers to a cell which has stably integrated foreign DNA into the genomic DNA.
The term xe2x80x9clong-term expressionxe2x80x9d as used herein means detectable expression for more than six (6) months and, more preferably, more than one (1) year, following transfection of cells in an immunocompetent (i.e., not immunocompromised) animal.
As used herein, the term xe2x80x9cgene of interestxe2x80x9d refers to a gene inserted into a vector or plasmid whose expression is desired in a host cell. Genes of interest include genes having therapeutic value as well as reporter genes. A variety of such genes are contemplated, including genes of interest encoding a protein which provides a therapeutic function. It is not intended that the present invention be limited to genes of interest encoding a particular protein having therapeutic function. A variety of such genes are contemplated including but not limited to the bilirubin UDP-glucuronosyltransterase gene, the dystrophin gene (which is capable of correcting the defect seen in the muscle of MD patients), the utrophin gene, the CFTR gene (capable of correcting the defect seen in cystitic fibrosis patients), the genes encoding enzymes (particular enzymes associated with enzyme deficiency diseases or diseases known to be caused by enzyme defects) and genes encoding clotting factors, angiogenesis factors, anti-angiogenesis factors, tumor suppressors, and suicide genes of which the Herpes thymidine kinase gene is an example. Genes of interest can be both endogenous and heterologous.
The term xe2x80x9creporter genexe2x80x9d indicates a gene sequence that encodes a reporter molecule (including an enzyme). A xe2x80x9creporter moleculexe2x80x9d is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. In one embodiment, the present invention contemplates the E. coli xcex2-galactosidase gene (available from Pharmacia Biotech, Pistacataway, N.J.), green fluorescent protein (GFP) (commercially available from Clontech, Palo Alto, Calif.), the human placental alkaline phosphatase gene, the chloramphenicol acetyltransferase (CAT) gene; other reporter genes are known to the art and may be employed (e.g., to easily follow success in transfection).
As used herein, the terms xe2x80x9cnucleic acid molecule encoding,xe2x80x9d xe2x80x9cDNA sequence encoding,xe2x80x9d and xe2x80x9cDNA encodingxe2x80x9d refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.
The present invention provides improved vectors, and in particular improved adenovirus vectors, for the delivery of recombinant genes to cells in vitro and in vivo. The description of the invention includes: A) selection of a ribosomal promoter; B) design of the expression vector; and C) delivery of the vector.
A. Selection of a Ribosomal Promoter
The present invention contemplates an expression vector comprising a ribosomal promoter sequence operably linked to a gene of interest. The invention is not limited by the nature of the ribosomal promoter sequence chosen; any non-viral promoter sequence or portion thereof which is functional in cells (i.e., such that a gene of interest can be expressed and/or overexpressed) may be utilized. A variety of ribosomal promoters are known to those skilled in the art.
The nucleotide sequences for such ribosomal promoters are known and available from the GenBank/EMBL database: Homo sapiens (X01547), Rattus norvegicus (X00677, K01588, M12030), Xenopus laevis (J01005), Xenopus borealis (X05263, Y00132, X00184), Drosophila melanogaster (X02210), Paracentrotus lividus (X63234), Tetrahymena pyrifornis (J01212, M10096), Dictyostelium discoideum (X00601), Arabidopsis thaliana (X15550), Pisum sativum (X52575), Triticum aestivum (X07841), Zea mays (X03990).
B. Design of the Vector
It is not intended that the present invention be limited to a particular vector. Both viral vectors and non-viral vectors are contemplated. In a preferred embodiment, adenovirus vectors are employed.
1. Adenovinrus Vectors
Self-propagating adenovirus (Ad) vectors have been extensively utilized to deliver foreign genes to a great variety of cell types in vitro and in vivo. xe2x80x9cSelf-propagating virusesxe2x80x9d are those which can be produced by transfection of a single piece of DNA (the recombinant viral genome) into a single packaging cell line to produce infectious virus; self-propagating viruses do not require the use of helper virus for propagation.
Existing Ad vectors have been shown to be problematic in vivo. This is due in part because current or first generation Ad vectors are deleted for only the early region 1 (E1) genes, as well as to the fact that viral regulatory units such as the CMV promoter/enhancer are inactivated over time. These vectors are crippled in their ability to replicate normally without the trans-complementation of E1 functions provided by human 293 cells, a packaging cell line [ATCC CRL 1573; Graham et al. (1977) J. Gen. Virol. 36:59]. Unfortunately, with the use of high titres of E1 deleted vectors, and the fact that there are E1-like factors present in many cell types, E1 deleted vectors can overcome the block to replication and express other viral gene products [Imperiale et al. (1984) Mol. Cell Biol. 4:867; Nevins (1981) Cell 26:213; and Gaynor and Berk (1983) Cell 33:683]. The expression of viral proteins in the infected target cells elicits a swift host immune response, that is largely T-cell mediated [Yang and Wilson (1995) J. Immunol. 155:2564 and Yang et al. (1994) Proc. Natl. Acad. Sci. USA 91:4407]. This immune response leads to changes in cytokine gene expression which can turn off transgenic expression when viral promoters are used. See L. Qin et al., xe2x80x9cPromoter Attention in Gene Therapy: Interferon-xcex3 and Tumor Necrosis Factor-xcex1 Inhibit Transgene Expression,xe2x80x9d Human Gene Therapy 8:2019 (1997).
The present invention contemplates adenovirus vectors employing novel endogenous promoter elements which are not inactivated by the immune response. Suitable adenovirus vectors are described in U.S. Pat. No. 5,585,362 to Wilson et al., entitled xe2x80x9cAdenovirus Vectors For Gene Therapy,xe2x80x9d hereby incorporated by reference. In one embodiment, the present invention contemplates a vector wherein mouse L32 ribosomal promoter sequences (FIG. 1) (SEQ ID NO:1) are used to regulate transgene expression, leading to persistence of expression for one year or more.
2. Other Viral Vectors
It is not intended that the present invention be limited to adenoviral vectors. A variety of other viral vectors are contemplated, including but not limited to lentiviral vectors, AAV (adeno-associated virus) vectors, retrovirus vectors, and herpes virus vectors.
C. Delivery of the Vector
Functional exogenous genes can be introduced to mammalian cells in vitro by a variety of physical methods, including transfection, direct microinjection, electroporation, and coprecipitation with calcium phosphate. While satisfactory for transfecting cells in culture, most of these techniques, however, are impractical for delivering genes to cells within intact animals. The present invention contemplates the use of viral vectors, as well as non-viral vectors for delivery of nucleic acid comprising a ribosomal promoter in operable combination with a gene of interest.
It is not intended that the present invention be limited to just one delivery approach. Exemplary delivery approaches are discussed below.
1. Receptor-Mediated Uncompacted DNA Delivery in Vivo
Receptor-mediated gene transfer has been shown to be successful in introducing transgenes into suitable recipient cells, both in vitro and in vivo. This procedure involves linking the DNA to a polycationic protein (usually poly-L-lysine) containing a covalently attached ligand, which is selected to target a specific receptor on the surface of the tissue of interest. The gene is taken up by the tissue, transported to the nucleus of the cell and expressed for varying times. The overall level of expression of the transgene in the target tissue is dependent on several factors: the stability of the DNA-carrier complex, the presence and number of specific receptors on the surface of the targeted cell, the receptor-carrier ligand interaction, endocytosis and transport of the complex to the nucleus, and the efficiency of gene transcription in the nuclei of the target cells.
Wu et al., U.S. Pat. No. 5,166,320 (hereby incorporated by reference), discloses tissue-specific delivery of DNA using a conjugate of a polynucleic acid binding agent (such as polylysine, polyarginine, polyornithine, histone, avidin, or protanine) and a tissue receptor-specific protein ligand. For targeting liver cells, Wu suggests xe2x80x9casialoglycoprotein (galactose-terminal) ligandsxe2x80x9d.
Wagner, et al., Proc. Natl. Acad. Sci., 88:4255-4259 (1991) and U.S. Pat. No. 5,354,844 (hereby incorporated by reference) disclose complexing a transferrin-polylysine conjugate with DNA for delivering DNA to cells via receptor mediated endocytosis. Wagner, et al., teach that it is important that there be sufficient polycation in the mixture to ensure compaction of plasmid DNA into toroidal structures of 80-100 nm diameter, which, they speculate, facilitate the endocytic event.
2. Direct Injection of Naked, Uncompacted DNA
The possibility of detecting gene expression by directly injecting naked DNA into animal tissues was demonstrated first by Dubenski et al., Proc. Nat. Acad. Sci. USA, 81:7529-33 (1984), who showed that viral or plasmid DNA injected into the liver or spleen of mice was expressed at detectable levels. The DNA was precipitated using calcium phosphate and injected together with hyaluronidase and collagenase. The transfected gene was shown to replicate in the liver of the host animal. Benvenisty and Reshef, Proc. Nat. Acad. Sci. USA, 83:9551-55 (1986) injected calcium phosphate precipitated DNA intraperitoneally into newborn rats and noted gene expression in the livers of the animals 48 hours after transfection. In 1990, Wolff et al., Science, 247:1456-68 (1990), reported that the direct injection of DNA or RNA expression vectors into the muscle of mice resulted in the detectable expression of the genes for periods for up to 2 months. This technique has been extended by Acsadi et al., New Biologist, 3:71-81 (1991) to include direct injection of naked DNA into rat hearts; the injected genes were expressed in the heart of the animals for up to 25 days. Other genes, including the gene for dystrophin have been injected into the muscle of mice using this technique. This procedure forms the base of a broad approach for the generation of immune response in an animal by the administration of a gene by direct injection into the target tissue. The gene is transiently expressed, producing a specific antigen. (See Donnelly et al., The Immunologist, 21, pp. 20-26 (1994) for a recent review). However, the DNA used in these experiments has not been modified or compacted to improve its survival in the cell, its uptake into the nucleus or its rate of transcription in the nucleus of the target cells.
3. Administration of Viral Vectors in the Portal Vein
In a particular embodiment, the genetic cassette is delivered to a particular tissue in said animal. It is not intended that the present invention be limited to the particular tissue type. In one embodiment, however, said tissue is selected from the group consisting of lung, trachea and liver tissue. For delivery to the liver, the hepatocytes can be readily transfected in vivo by direct vector infusion in the portal vein, as well as to the peripheral circulation.
One hepatic metabolic deficiency that has attracted broad interest due to the hope that it might be cured by gene therapy is Crigler-Najjar syndrome type I, familial non-hemolytic jaundice with kernicterus. Crigler-Najjar syndrome is an autosomal recessive inherited metabolic defect in bilirubin glucuronosyltransferase activity. Patients with Crigler-Najjar syndrome type I do not conjugate bilirubin and a striking unconjugated hyperbilirubinemia develops, leading to kernicterus and death, usually in infancy or childhood. Patients with one normal allele have an essentially normal phenotype suggesting that partial genetic correction may lead to a functional cure in Crigler-Najjar type I patients. The usual treatments for Crigler-Najjar syndrome, phototherapy and pharmacologic treatment, are not adequate to keep patients with type I disease alive. Crigler-Najjar syndrome type I patients theoretically are ideal candidates for liver directed human gene therapy trials as complementation of a genetic enzymatic defect has the theoretic potential to cure disease before the sequela of kernicterus leads to death.
The human bilirubin UDP-glucuronosyltransferase gene locus (UGT1) encodes multiple glucuronosyltransferases generated by alternately splicing variable 5xe2x80x2 introns (and the corresponding exons) with a common 3xe2x80x2 region consisting of exons 2-5. Complementation of the genetic deficiency in primary and immortalized Gunn rat hepatocytes with retroviruses and plasmids has demonstrated the ability of HUG Br 1 and HUG Br 2 vectors to express bilirubin UDP-glucuronosyltransferase activity in these cells. Human fibroblasts derived from a Crigler-Najjar patient have also been shown to express bilirubin glucuronosyltransferase activity following HUG Br 1 gene transfer. The presence of multiple bilirubin UDP-glucuronosyltransferase isoforms raised the question as to whether gene transfer of a single isoform could correct the Crigler-Najjar phenotype in vivo.
One feature which makes Crigler-Najjar syndrome type I attractive to study is the availability of an authentic animal model of the human disease, the Gunn rat. The majority of bilirubin UDP-glucuronosyltransferase activity is normally expressed in the liver, and hepatocytes are therefore a logical target to treat this disease. The present invention describes in vivo adenovirus-mediated gene transfer of the genetic cassette encoding the major human bilirubin UDP-glucuronosyltransferase isoform, HUG Br 1, to complement the genetic deficiency in Gunn rat hepatocytes.